Morphogenesis and remodeling of bone are accomplished by the coordinated actions of bone-resorbing osteoclasts and bone-forming osteoblasts, which metabolize and remodel bone structure throughout development and adult life. Bone is constantly being resorbed and formed at specific sites in the skeleton called basic multicellular units. An estimated 10% of the total bone mass in the human body is remodeled each year. Upon activation, osteoclasts, which differentiate from hematopoietic monocyte/macrophage precursors, migrate to the basic multicellular unit, resorb a portion of bone and finally undergo apoptosis. Subsequently, newly generated osteoblasts, arising from preosteoblastic/stromal cells, form bone at the site of resorption. The development of osteoclasts is controlled by preosteoblastic cells, so that the processes of bone resorption and formation are tightly coordinated, thus allowing for a wave of bone formation to follow each cycle of bone resorption. Imbalances between osteoclast and osteoblast activities can result in skeletal abnormalities characterized by decreased (osteoporosis) or increased (osteopetrosis) bone mass (Khosla, Endocrinology, 2001, 142, 5050-5055; Nakashima et al., Curr. Opin. Rheumatol., 2003, 15, 280-287).
Communication between osteoblasts and osteoclasts occurs through cytokines and cell-to-cell contacts. A cytokine that performs a key regulatory role in bone remodeling is receptor activator of NF-kappaB ligand (RANKL). RANKL was first identified as a tumor necrosis factor (TNF) superfamily member [also known as tumor necrosis-factor-related activation-induced cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation factor (ODF) and tumor necrosis factor (ligand) superfamily member 11 (TNFSF11)] and was subsequently identified as a factor that is capable of inducing osteoclast differentiation in vitro (Anderson et al., Nature, 1997, 390, 175-179; Lacey et al., Cell, 1998, 93, 165-176; Wong et al., J. Exp. Med., 1997, 186, 2075-2080; Yasuda et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 3597-3602). The human RANKL gene maps to chromosome 13q14. The highest expression levels of RANKL are found in bone, bone marrow and lymphoid tissues (Anderson et al., Nature, 1997, 390, 175-179; Lacey et al., Cell, 1998, 93, 165-176; Wong et al., J. Exp. Med., 1997, 186, 2075-2080; Yasuda et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 3597-3602) and it can also be detected in brain, heart, kidney, skeletal muscle and skin (Kartsogiannis et al., Bone, 1999, 25, 525-534).
RANKL is assembled from three RANKL subunits to form the functional trimeric molecule. RANKL is initially anchored to the cell membrane, and a small fraction of the protein released from the cell surface by the proteolytic action of the metalloprotease-disintegrin TNF-alpha convertase (TACE) (Lum et al., J. Biol. Chem., 1999, 274, 13613-13618). RANKL is both necessary and sufficient to stimulate of osteoclast differentiation and activity as well as to inhibit osteoclast apoptosis (Fuller et al., J. Exp. Med., 1998, 188, 997-1001; Lacey et al., Cell, 1998, 93, 165-176; Lum et al., J. Biol. Chem., 1999, 274, 13613-13618; Yasuda et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 3597-3602). RANKL is expressed on the surface of preosteoblastic and bone marrow stromal cells. Its expression can be positively or negatively modulated by various hormones, cytokines, growth factors and glucocorticoids, including, vitamin-D3, parathyroid hormone (PTH), interleukin 1-beta and TNF-alpha, all of which increase RANKL expression (Kong et al., Immunol. Today, 2000, 21, 495-502).
At the initiation of the cycle of bone resorption and formation, RANKL binds to its functional receptor RANK on preosteoclastic cells (Anderson et al., Nature, 1997, 390, 175-179; Lacey et al., Cell, 1998, 93, 165-176). This interaction between RANKL and RANK stimulates the formation of mature osteoclasts, which are phenotypically characterized by multinucleation, bone-resorbing function and expression of the lineage specific marker tartrate-resistant acid phosphatase (TRAP) (Burgess et al., J. Cell. Biol., 1999, 145, 527-538; Hsu et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 3540-3545; Lum et al., J. Biol. Chem., 1999, 274, 13613-13618; Yasuda et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 3597-3602). Alternatively, RANKL can bind to the soluble receptor osteoprotegerin (OPG), which is expressed primarily by bone marrow stromal cells and serves to inhibit osteoclast maturation and activation by RANKL (Lacey et al., Cell, 1998, 93, 165-176; Yasuda et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 3597-3602). PTH, a major regulator of bone remodeling, stimulates osteoclast function by simultaneously increasing RANKL expression while decreasing OPG expression (Lee and Lorenzo, Endocrinology, 1999, 140, 3552-3561). As preosteoblastic cells differentiate, RANKL mRNA levels are significantly reduced, whereas OPG mRNA levels increase (Gori et al., Endocrinology, 2000, 141, 4768-4776). Such a dynamic relationship between RANKL and OPG levels allows for a wave of osteoclast activity to be followed by a wave osteoblast activity, thereby completing the cycle of bone resorption and formation.
RANKL induces a transient elevation of calcium in osteoclasts due to release of calcium from intracellular stores (Komarova et al., J. Biol. Chem., 2003, 278, 8286-8293). In T-cells, T-cell receptor activation-induced calcium mobilization is solely responsible for the induction of RANKL expression (Wang et al., Eur. J. Immunol., 2002, 32, 1090-1098).
Mice homozygous for disruption of the RANKL gene are born at the expected frequency, but show severely retarded growth after weaning at three weeks of age. RANKL deficient mice exhibit severe osteopetrosis (thickening of bone), defects in tooth eruption and a complete lack of osteoclasts due to the inability of osteoblasts to support osteoclastogenesis (Kong et al., Immunol. Today, 2000, 21, 495-502).
RANKL function is not restricted to bone morphogenesis and remodeling. RANKL-deficient mice also display defects in early differentiation of T- and B-lymphocytes and lack all lymph nodes, demonstrating that RANKL is a regulator of lymph-node organogenesis and lymphocyte development, in addition to being an essential osteoclast differentiation factor (Kong et al., Immunol. Today, 2000, 21, 495-502). T-cell receptor stimulation induces RANKL gene expression, which subsequently leads to activation of c-Jun N-terminal kinase in T-cells (Wong et al., J. Biol. Chem., 1997, 272, 25190-25194). RANKL also participates in immune system function as an important survival factor for bone marrow derived dendritic cells by inhibiting apoptosis in these cells (Lum et al., J. Biol. Chem., 1999, 274, 13613-13618; Wong et al., J. Exp. Med., 1997, 186, 2075-2080). Additionally, RANKL is also required for the development of lobulo-alveolar mammary structures during pregnancy in mice (Fata et al., Cell, 2000, 103, 41-50).
Inappropriate activation of osteoclasts by RANKL can create an imbalance between the processes of bone resorption, resulting in the rate of bone resorption exceeding that of bone formation. Local or generalized bone loss is observed in many osteopenic disorders, including postmenopausal and age-related osteoporosis, periodontitis, familial expansile osteolysis and Paget's disease (Khosla, Endocrinology, 2001, 142, 5050-5055). Upregulation of RANKL mRNA has been reported in several of these diseases.
Paget's disease is characterized by large numbers of abnormal osteoclasts that induce increased bone resorption. RANKL mRNA expression is elevated in both cell lines and bone marrow derived from patients with Paget's disease. Furthermore, osteoclast precursors from Paget's disease patients undergo osteoclastogenesis at a much lower concentration of RANKL than normal cells (Menaa et al., J. Clin. Invest., 2000, 105, 1833-1838).
Other diseases with osteopenic pathologies, such as rheumatoid arthritis, chronic viral infection and adult and child leukemias, are characterized by activated T-cells and bone destruction (Kong et al., Immunol. Today, 2000, 21, 495-502). Rheumatoid arthritis is a chronic inflammatory disease characterized by progressive osteoclast-mediated bone resorption. Rheumatoid arthritis synovial fluid contains osteoclast precursors, RANKL-expressing T-cells and OPG-producing B-cells. Cultured macrophages from rheumatoid arthritis synovial fluid can differentiate into osteoclasts in a RANKL-dependent process (Itonaga et al., J. Pathol., 2000, 192, 97-104). In a T-cell dependent rat model of experimentally-induced arthritis that mimics many of the clinical features of human rheumatoid arthritis, inhibition of RANKL function through OPG treatment prevents bone destruction (Kong et al., Nature, 1999, 402, 304-309).
Multiple myeloma is a cancer in which osteoporosis and bone destruction are prominent features. Myeloma cell lines stimulate RANKL expression while inhibiting OPG expression by bone marrow stromal cells, resulting in a disruption of the balance between RANKL and OPG levels, followed by the aberrant production and activation of osteoclasts (Pearse et al., Proc. Natl. Acad. Sci. USA, 2001, 98, 11581-11586). A secreted form of RANKL is also expressed by cancer cells responsible for humoral hypercalcemia of malignancy (Nagai et al., Biochem. Biophys. Res. Commun., 2000, 269, 532-536). An increase in RANKL with a concurrent decrease in OPG expression is also observed following glucocorticoid treatment of osteoblastic lineage cells, which also stimulates osteoclastogenesis of these cells, suggesting a mechanism by which systemic glucocorticoid use leads to severe osteoporosis (Hofbauer et al., Endocrinology, 1999, 140, 4382-4389).
These findings demonstrate a link between immune function and bone physiology and also provide a molecular explanation for bone density loss associated with immune disorders and suggest that inhibition of RANKL function, and consequently osteoclast activity, can ameliorate osteopenic conditions (Kong et al., Immunol. Today, 2000, 21, 495-502).
Disclosed and claimed in the PCT publication WO 99/29865 are a nucleic acid molecule encoding human RANKL, antibodies that recognize RANKL polypeptides and cells modified to increase expression of RANKL polypeptides. Also disclosed and claimed is the modulation of an immune response in a mammal, wherein the modulator can be selected from a group consisting of antisense RANKL nucleic acid comprising at least one phosphodiester bond (Choi et al., 1999).
Disclosed in the U.S. Pat. No. 6,017,729 is a method for the isolation of a nucleic acid molecule encoding human RANKL, the nucleotide sequence encoding human RANKL and a method for the preparation of monoclonal antibodies that recognize RANKL (Anderson et al., 2000).
Disclosed and claimed in the PCT publication WO 01/53486 is a nucleic acid molecule having at least 80% nucleotide identity to a nucleic acid sequence encoding human RANKL, an antibody that binds to the polypeptide sequence encoded by a human RANKL nucleic acid molecule and the method of producing said antibody. Also disclosed and claimed is a method of inhibiting tumor cell growth by exposing tumor cells to an agent that inhibits the expression of polypeptide encoded by a human RANKL nucleic acid molecule, wherein the agent can be an antisense oligonucleotide that hybridizes to a RANKL nucleic acid molecule (Ashkenazi et al., 2001).
The PCT publication WO 01/23559 discloses and claims the nucleotide sequence containing a regulatory region of the human RANKL gene, as well as a method for identifying an antagonist to inhibit RANKL expression (Chandrasekhar et al., 2001).
The U.S. Pat. No. 6,271,349 discloses a method for the isolation of a nucleic acid molecule encoding RANKL, and a nucleotide sequence encoding human RANKL (Dougall and Galibert, 2001).
Disclosed and claimed in the U.S. Pat. No. 6,242,213 are a nucleic acid molecule encoding human RANKL, a host cell transformed or transfected with said nucleic acid molecule and a process of preparing RANKL polypeptide molecules. This publication also discloses that useful fragments of a RANKL nucleic acid molecule can include antisense or sense oligonucleotides (Anderson, 2001).
Disclosed and claimed in the U.S. pre-grant publication 20020159970 are a nucleic acid molecule encoding human RANKL, antibodies that recognize RANKL polypeptides and cells modified to increase expression of RANKL polypeptides. Also disclosed and claimed is a modulation of an immune response in a mammal, wherein the modulator can be selected from a group consisting of an antisense RANKL nucleic acid comprising at least one phosphodiester bond (Choi et al., 2002).
Disclosed the PCT publication WO 02/16551 are methods for treating a mammal having a disorder with a RANKL-inhibiting agent that is an antisense nucleic acid directed against RANKL RNA or with a RANKL-increasing agent that is a polypeptide (Choi et al., 2002).
The U.S. pre-grant publication 20020182586 discloses a method treating individuals with carcinoma cancer inhibitors which include antisense molecules that target a nucleic acid molecule encoding RANKL (Morris and Engelhard, 2002).
The U.S. pre-grant publication 20030017151 and the PCT publication WO 02/092016 disclose and claim a method of treating a patient with RANKL antisense oligonucleotides, an antibody that binds to a RANKL polypeptide or a ribozyme that cleaves RANKL mRNA to stimulate bone formation (Dougall and Anderson, 2003).
The U.S. pre-grant publication 20030021785 and the PCT publication WO 02/098362 disclose the use of an antisense approach to target RANKL mRNA transcripts (Dougall, 2003).
The Japanese patent application JP 2001157587 claims and discloses a nucleic acid sequences encoding human RANKL (Nagai, 2001).
As a consequence of RANKL involvement in many diseases, there remains a long felt need for additional agents capable of effectively regulating RANKL function. As such, inhibition is especially important in the treatment of disorders characterized by bone destruction, given that the upregulation of expression of RANKL is associated with so many different types of osteopenic diseases.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and has been proven to be uniquely useful in a number of therapeutic, diagnostic, and research applications.
The present invention provides compositions and methods for modulating RANKL expression, and consequently RANKL function.